Normal mammalian cells have a finite capacity for division in culture. After completing their in vitro lifespan they enter a non-dividing state that has been termed cellular senescence. This phenomenon has been most extensively studied in normal human diploid fibroblasts. A number of changes in gene expression have been observed when cells become senescent. However, it has not been possible to determine which changes are the cause as distinct from the result of cellular senescence. Cell fusion studies have clearly demonstrated that the senescent phenotype is dominant and that immortal cells result from recessive changes in the normal cell program. Evidence has accumulated over the past several years that senescent cells produce an inhibitor(s) of DNA synthesis. One such DNA synthesis inhibitor (SDI-1) was cloned from a senescent cell cDNA library, and was the first of a group of Cdk inhibitors to be discovered. Subsequently, six Cdk inhibitors have been cloned, many of which can inhibit DNA synthesis when over-expressed in mammalian cells. The mRNA level of Sdi1 (p21) is over-expressed 10-20 fold in senescent compared with young human diploid fibroblasts. We have found that decreasing the level of p21, following induction of antisense p21 sequences, results in stimulation of density inhibited, young human fibroblasts to undergo DNA synthesis and divide. However, expression of antisense p21 in senescent human cells is not sufficient to cause them to enter S phase. Therefore, we hypothesize that the inability of senescent cells to synthesize DNA may be due to the over-expression of multiple Cdk inhibitors, and the goal of this project is to determine the role of Cdk inhibitors in cellular senescence.